The gypsy moth, Lymantria dispar, is a particularly troublesome tree defoliator. Since its import into the United States in 1869 for use in the production of silk fibers in Massachusetts, the gypsy moth has caused major tree infestations. The gypsy moth has spread throughout New England, New York, Delaware, Maryland, New Jersey, Pennsylvania, Virginia, West Virginia, Ohio, Michigan, North Carolina, and Wisconsin. (McFadden, et al., Forest Insect Guilds: Patterns of Interaction with Host Trees, Y. N. Baranchikov, et al., pp. 172-186, U.S. Department of Agriculture, For. Serv. Gen. Tech. Rep. NE-153, Radnor, Pa., 1989; Blackburn, et al. Proceedings of the Annual Gypsy Moth Review, pp. 261-351, 1994).
Several chemical pesticides have been used in the control of gypsy moth infestations. DDT is one of the most effective chemical insecticides in controlling gypsy moths. However, DDT and other chemical insecticides have been found to adversely affect the environment in myriad ways. Broad spectrum insecticides such as DDT are not only effective in exterminating unwanted pests; these chemicals also kill insects that we consider beneficial. Destruction of insect species results in disruptions to the entire ecosystem. For control of gypsy moth infestations, DDT use has been abandoned in favor of a less harmful chemical, diflubenzeron, and the biorational agent Bacillus thuringiensis (Bt).
A growing awareness that forestry and agricultural practices have a great impact on the environment has spawned research directed toward the development of biological insect control agents as an alternative to the use of chemical pesticides. Insect baculoviruses have received considerable attention as promising agents for the biological control of insects. These viruses have been isolated from hundreds of insect species, have a narrow host range, can be formulated for aerial application, and can be manipulated through genetic engineering to enhance viral efficacy. Insect baculoviruses have essentially no adverse environmental impact.
Baculoviruses, such as the Lymantria dispar nuclear polyhedrosis virus (LdNPV), have two distinct morphological forms which serve different roles in the process of infection (Blissard and Rohrmann, Annu. Rev. Entomol., 35:127-155, 1990). The infection cycle begins with larval ingestion of a polyhedron, a polyhedral shaped crystalline protein matrix that contains viral particles. The polyhedron is dissolved within the insect midgut, thereby releasing the occluded virus particles, which in turn infect the insect. Early after infection, a budded (nonoccluded) form of virus is produced in infected cells. Budded virions cause a systemic infection of the insect larval host. Late in infection, virions that are produced become occluded into polyhedra.
A gypsy moth virus, LdNPV, has been used on a limited basis as a gypsy moth control agent. One advantage of LdNPV is that it is specific for the gypsy moth. Consequently, LdNPV is the agent of choice for gypsy moth control, particularly in environmentally sensitive areas. However, control of gypsy moth by LdNPV has not been fully exploited due to high production costs and low efficacy.
There is currently no commercial production of LdNPV. LdNPV is produced in limited amounts by the Forest Service and the Animal and Plant Health Inspection Service in gypsy moth larvae. Production in larvae is expensive; currently, the cost of generating enough virus to treat an acre of forest with the viral insecticide is several times higher than treatment with diflubenzeron or Bt.
Production of LdNPV in cell culture is a less expensive alternative to production in larvae. The virus may be propagated in cell culture using the L. dispar 652Y (Ld652Y) ovarian cell line. However, propagation of nuclear polyhedrosis viruses in cell culture generally yields low numbers of polyhedra, the protein-encapsulated form of the virus that is most suitable for use as an insecticide. Nuclear polyhedrosis viruses tend to mutate to a form that produces few polyhedra upon serial transfer, a process that is necessary during scale-up of cell cultures. Baculovirus few polyhedra (FP) mutants arise at a high frequency during serial passage in cell culture. In contrast to wild-type or many polyhedra (MP) virus, FP mutants produce few polyhedra, and those that are produced are essentially noninfectious. Lack of potency is probably due to a reduced number of viral particles found in the polyhedra of FP mutants (Hink and Strauss, J. Invertebr. Pathol., 27:49-55, 1976; Potter, et al., J. Virol., 18:1040-1050, 1976; Fraser and Hink, Virology, 177:366-378, 1982; Slavicek et al., Biol. Con., 5:251-261, 1995). FP mutants also synthesize greater numbers of budded virus than wild-type virus. Increased production of budded virus results in the conversion of the virus population from one exhibiting an MP phenotype to one with an FP phenotype during serial passage in cell culture. FP mutants have been identified in the Autographa californica MNPV (Hink and Strauss, J. Invertebr. Pathol., 27:49-55, 1976), Trichoplusia ni MNPV (Mackinnon, et al., J. Ultrastruc. Res., 49:419-435, 1974; Potter, et al., J. Virol., 18:1040-1050, 1976), Galleria mellonella MNPV (Fraser and Hink, Virology 177:366-378, 1982), and LdNPV (Slavicek, et al., J. Invertebr. Pathol., 59:142-148, 1992; Slavicek, et al., Biol. Con. 5:251-261, 1995). Because the efficacy of a viral insecticide is a function of the concentration of infective viral particles, the formation of mutants that produce fewer polyhedra (FP mutants) reduces the efficacy and increases the cost of viral insecticides.
One means by which the efficacy of the viral insecticide may be increased and the per acre cost of viral pest control reduced is by increasing the number of polyhedra produced in cell culture. That may be accomplished by identifying and employing a viral strain that produces large numbers of polyhedra in cell culture, and which does not mutate into the FP form upon serial transfer. U.S. Pat. No. 5,420,031, incorporated by reference herein, discloses a strain of LdNPV (A21-MPV) (ATTC VR2396) that exhibits enhanced polyhedra production stability. This strain has a low frequency of FP mutants relative to wild-type and produces polyhedra at fairly consistent levels. U.S. Pat. No. 5,462,732, incorporated by reference herein, discloses a method for protecting plants from insects against which LdNPV is toxic using ATCC VR2396. Although cells infected with ATCC VR2396 produce higher yields of polyhedra, the viral insecticide continues to be a costly alternative to other, less environmentally friendly, methods of controlling gypsy moth.
In addition to polyhedra yields, other factors that can contribute to the cost of production of viruses in cell culture include the multiplicity of infection needed to achieve infection of the cells and the number of passages that are needed during scale up to large bioreactors. What is needed in the art is a strain of LdNPV that can infect cultured cells at a lower multiplicity of infection, that requires fewer passages, and that produces greater numbers of polyhedra than can be achieved with strains that are known to the art.